Method for monitoring early treatment response

ABSTRACT

Disclosed is a method for monitoring early treatment response of a cancer treatment comprising measuring by magnetic resonance spectroscopy (MRS), for example, proton MRS, the amount of Choline present in the tissue adjoining or surrounding the cancerous tissue before and after treatment; the treatment comprises administration of an angiogenesis inhibitor, for example, a VEGF inhibitor, whereby a decrease in the amount of Choline after treatment is indicative of a positive response. The decrease in the amount of Choline represents the decrease in the internal cell membrane as a result of down regulation of the organelles and their secretory granules and their transport vesicles. Disclosed also is a method for determining effectiveness of an angiogenesis inhibitor in the treatment of cancer. Also disclosed are methods of monitoring early treatment response in diseases where an angiogenesis effector, i.e., an inhibitor or promoter of angiogenesis, is employed.

FIELD OF THE INVENTION

This invention pertains to a method for monitoring early response to atreatment of a disease in general, and cancer in particular, forexample, a cancer treatment involving the use of an angiogenesisinhibitor. The method employs Magnetic Resonance Spectroscopy (MRS).

BACKGROUND OF THE INVENTION

There is a need for monitoring the effectiveness of a disease treatment,particularly for diseases where the condition of the patient candeteriorate rapidly if the treatment is not effective. One such diseaseis cancer, particularly, metastasis of cancer, which involvesangiogenesis. Angiogenesis, which is formation of new blood vessels frompreexisting ones, is known to be important for tumor growth andmetastasis. Without blood supply, tumor growth is diffusion-limited, andis generally restricted to less than 1 mm³ to 2 mm³. The recruitment ofa vascular supply allows a tumor to grow beyond this limited volume andprovides a route for metastasis. Angiogenesis supplies oxygen andnutriments needed for tumor growth.

Typically, the angiogenesis process takes place as follows. The diseasedtissue produces and releases angiogenic growth factors (proteins) thatdiffuse into the nearby tissues. The angiogenic growth factors bind tospecific receptors located on the endothelial cells of nearbypreexisting blood vessels. Once growth factors bind to their receptors,the endothelial cells become activated. Signals are sent from the cell'ssurface to the nucleus. The endothelial cell's machinery begins toproduce new molecules including enzymes. Enzymes form tiny openings inthe sheath-like covering (basement membrane) surrounding all existingblood vessels. The endothelial cells begin to divide (proliferate), andthey migrate out through the openings of the existing vessel towards thediseased tissue (tumor). Specialized molecules called adhesionmolecules, or integrins (α_(v)β₃, α_(v)β₅), serve as grappling hooks tohelp pull the sprouting new blood vessel sprout forward. Additionalenzymes (matrix metalloproteinases or MMP) are produced to dissolve thetissue in front of the sprouting vessel tip in order to accommodate it.As the vessel extends, the tissue is remolded around the vessel.Sprouting endothelial cells roll up to form a blood vessel tube.Individual blood vessel tubes connect to form blood vessel loops thatcan circulate blood. The newly formed blood vessel tubes are stabilizedby specialized muscle cells (smooth muscle cells, pericytes) thatprovide structural support. Blood flow begins.

In angiogenesis, the neoplastic cells can recruit, in addition to normalendothelial cells, the macrophages, fibroblasts, mast cells, and/orplatelets to generate new vessels from pre-existing ones at theperiphery of the tumor. Angiogenesis is thus orchestrated by a web ofsignaling pathways controlled by proangiogenic protein signalingmolecules. The proangiogenic signaling molecules arise from the tumorand react with the cells in the surrounding normal tissue (paracrinemolecules). The proangiogenic signaling molecules are generated fromcells in the normal surrounding tissue reacting with adjacent,non-neoplastic cells, and reacting with matrix of the tissue (juxtacrinemolecules). Proangiogenic signaling proteins are generated from a cellin the tissue that reacts with itself (autocrine molecules). Fisher, M.J., et al., Neuroimg. Clin. N. Am. 12, 477-499 (2002), and Ch. 9 inMolecular Basis of Medical Cell Biology, 1st ed., Fuller, G. M., et al.(Eds.) Appleton and Large (1998).

Some of the common approaches to cancer treatment (including metastasisof cancer) involve surgery, radiation therapy, and/or chemotherapy.Radiation therapy and chemotherapy are effective if they are capable ofkilling the tumor cells; i.e., when they act as cytotoxic agents.Typically, the response to radiation therapy or chemotherapy ismonitored by magnetic resonance imaging (MRI) of the tumor, wherein adecrease in tumor size is indicative of positive response to treatment.

Angiogenesis inhibitors have been proposed for cancer treatment. Forexample, interrupting the signaling proangiogenic pathways interruptsthe blood supply to the tumor, and thereby, stops neoplasticproliferation. The major proangiogenic signaling pathways involve growthfactors, integrins/proteases, coagulation/fibrinolysis factors andinflammatory factors.

The angiogenesis inhibitors are cytostatic rather than cytotoxic;accordingly, classical signs of treatment response, e.g., decreasedtumor size or decreased enhancement, commonly observed in treatmentsinvolving cytotoxic agents, may not be observed with cytostaticangiogenesis inhibitors. Accordingly, classical imaging techniques suchas MRI alone may not be suitable or adequate to monitor response to atreatment involving angiogenic inhibitors.

Magnetic Resonance Spectroscopy or MRS has been proposed as a tool forobtaining information on cellular metabolism; see, for example, Norfray,J. et al., Ch. 110 in Pediatric Neurosurgery, 4^(th) ed., McLone, D. G.,et al. (Eds), W.B. Saunders Co. (2001). MRS also has been proposed fordiagnosing the treatment response of tumors with cytotoxic agents; see,for example, Fulham, M. J., et al., Radiology, 185, 675-686 (1992),which discloses that brain tumor metabolism was studied with ¹H MRSbefore and after treatment with radiation therapy. MRS permitsnon-invasive examination of metabolic characteristics of human cancersin a clinical environment. Accessible nuclei include ³¹P, ¹³C, ¹H, and²³Na. ³¹P MRS contains information about energy status (phosphocreatine,inorganic phosphate, and nucleoside triphosphates), phospholipidsmetabolites (phosphomonoesters and phosphodiesters), intracellular pH(pH NMR), and free cellular magnesium concentration (Mg²⁺ f).Water-suppressed ¹H MRS shows total choline, total creatine, lipids,glutamate, inositols, lactate, and the like. Negendank, W., NMR inBiomedicine, 5, 303-324 (1992).

Further, U.S. Pat. No. 6,681,132 (Katz et al.) discloses a method fordetermining the effectiveness of chemotherapy comprising administering adose of a cytotoxic antineoplastic agent to a subject prior to surgicalremoval of a cancerous tumor, acquiring magnetic resonance data from thesubject, and determining whether the treatment has affected thepopulation of a nucleus or nuclei, particularly ²³Na. Negendank, W.,supra, provides a review of various studies of human tumors by MRS.

In addition, Ross, B. et al., The Lancet, 641-646 (1984) disclosesmonitoring response to cytotoxic chemotherapy of intact human tumors by³¹P MRS; Griffiths, J. R. et al., The Lancet, 1435-36 (1983) disclosesthe use of ³¹P MRS to follow the progress of a human tumor duringchemotherapy with doxorubicin; Ross, B. et al., Arch. Surg., 122,1464-69 (1987) discloses the monitoring of chemotherapeutic treatmentresponse of osteosarcoma and other neoplasms of the bone by ³¹P MRS; andNorfray, J. F. et al., J. Computer Assisted Tomography, 23(6), 994-1003(1999) discloses an MRS study of the neurofibromatosis type 1intracranial lesions.

While MRS is effective as a tool for monitoring treatment response, thedisclosures in the art show that it has been applied to monitor theresponse to cytotoxic agents (radiation and chemotherapy). In manycases, a detectable change in tumor size is observed only after asignificantly long period of time, for example, after treatment for aperiod of about 3 months or more. Such long periods of time could beharmful to the patient, especially if the treatment has not beeneffective or only partially effective, such as, for example, treatmentsinvolving the use of angiogenesis inhibitors; during this long period oftime, tumor cells could multiply or metastasize, and lead to worseningof the patient's condition.

The foregoing shows that there exists a need for a method where an earlytreatment response can be monitored in diseases, especially where thetreatment involves the use of one or more angiogenesis inhibitors.Accordingly, the present invention provides such a method. This andother advantages of the invention, as well as additional inventivefeatures, will be apparent from the description of the inventionprovided herein.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method for monitoring early treatment responseof a disease treatment, particularly cancer treatment, comprisingmeasuring by MRS, the amount of Choline, e.g., total choline, present ina tissue adjoining or surrounding the diseased or cancerous tissuebefore and after treatment. The treatment, in one aspect, comprisesadministration of an angiogenesis inhibitor, whereby a decrease in theamount of Choline after treatment is indicative of a positive response.In accordance with the invention, the tissue surrounding the tumor canbe monitored by following cell membrane metabolism utilizing the Cholinepeak on ¹H MR spectroscopy. The Choline peak represents the visiblemobile Choline forming the plasma and organelle cell membranes. Adecrease in the Choline identifies treatment response; an increase inthe Choline peak identifies treatment failure.

The present invention also provides a method for monitoring earlytreatment response of an angiogenesis effector, i.e., an inhibitor orpromoter of angiogenesis, treatment in a diseased animal comprisingmeasuring, by Magnetic Resonance Spectroscopy (MRS), the amount ofCholine present in the diseased tissue before and after treatment,wherein said treatment comprises administration of an angiogenesiseffector, whereby a change in the amount of Choline after treatment isindicative of a response.

The present invention provides one or more advantages, for example,changes in the amount of Choline occurring immediately following atreatment can be detected by the method of the present invention evenbefore the amount of change required for determination by classicalmethods such as MRI imaging occurs in the tissue. When MRS is used inconjunction with MRI, in accordance with an embodiment of the invention,the present invention offers the combined advantages of MRI and MRS andprovides a method to monitor early treatment response. The presentinvention also provides a method for monitoring cancer treatment,especially a treatment of cancer susceptible to metastasize.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically depicts an axial view of a human brain 1. A regionof interest (ROI) is depicted as rectangle 2 comprising the tumor 8.Voxels 3-6 are the areas for determining the amounts of Choline in thecell membranes. 3 is V(T), a voxel located within the tumor. 4 is V(−A),a voxel where angiogenesis does not take place. 5 is V(+A), a voxelwhere angiogenesis takes place. 6 is V(N), a voxel in the normal tissue.7 depicts lateral ventricles.

FIG. 2 depicts an illustration of ¹H MRS peaks corresponding to Choline(CHO) and creatine (CRE) in voxels V(N), V(+A), V(−A) and V(T). TheX-axis represents chemical shift in parts per million (ppm) and theY-axis represents peak intensities of the metabolites in arbitraryunits. The CHO/CRE ratio is illustrated for each voxel.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated on monitoring changes in the amountof one or more metabolites occurring in an internal cell membrane, forexample, changes induced by the down-regulation of one or more of theintracellular organelles and their secretory granules and transportvesicles. The internal cell membranes constitute nearly 90% of the totalcell membranes, and form the membranes of the nucleus, the mitochondria,the lysosomes, the peroxisomes, the endoplasmic reticulums, the Golgiapparatus, the secretory granules, and the transport vesicles. Theangiogenesis inhibitors down-regulate the intracellular organelles andtheir secretory granules and transport vesicles of cells recruited bythe tumor at its periphery.

Accordingly, the present invention provides, in an embodiment, a methodfor monitoring early treatment response of a cancer treatment comprisingmeasuring, by Magnetic Resonance Spectroscopy (MRS), the amount ofCholine present in a tissue adjoining or surrounding the canceroustissue before and after treatment; the treatment comprisesadministration of an angiogenesis inhibitor, whereby a decrease in theamount of Choline after treatment is indicative of a positive response.MRS can be based on the resonance of any suitable nuclei; for example,nuclei selected from the group consisting of ³¹P, ¹H, ¹³C, and ²³Na, andany combination thereof; preferably, ¹H.

The term “Choline” herein is used to denote choline ((CH₃)₃N⁺CH₂CH₂OH),a derivative of choline, or a combination of choline and/or one or morederivatives of choline. Examples of choline derivatives includelysophosphatidylcholine, or glycerophosphocholine, phosphomonoesters ofcholine (e.g., phosphocholine), phosphodiesters of choline (e.g.,phosphatidylcholine), sphingomyelin, phosphoethanolamine,glycerophosphoethanolamine, or any combination thereof. In an embodimentof the invention, the term “Choline” represents the sum of choline andall choline derivatives (or total choline), for example, the sum ofcholine and phosphocholine. Phosphoserine and glycerophosphoserine alsocan be monitored Ruiz-Cabello, J. et al., NMR in Biomedicine, 5, 223-233(1992); Podo, F., NMR in Biomedicine, 12, 413-439 (1999); and Blüms, S.et al., Magn. Reson. Med., 42, 643-654 (1999).

A decrease in the amount of Choline occurs very early in the surroundingcells recruited by the tumor as a result of the down-regulation. Sincecholine contains 9 protons per molecule (as trimethylamines ortrimethylammonium salts), the Choline signal is amplified nine-fold.Since up to 90% of the cell membranes can be down-regulated in recruitedcells, MRS provides a sensitive method to monitor early treatmentresponse.

The amount of Choline can be measured by MRS in any suitable manner. Forexample, the amount of Choline can be measured by measuring the heightof a peak or peaks corresponding to Choline. In another embodiment, theamount of Choline can be measured by measuring the area under a peak orpeaks corresponding to Choline. In yet another embodiment, the amount ofCholine can be measured by measuring the ratio of the height of a peakor peaks corresponding to Choline relative to the height of a peak orpeaks of an internal standard. In a further embodiment, the amount ofCholine can be measured by measuring the ratio of the area under a peakor peaks corresponding to Choline relative to the area under a peak orpeaks of an internal standard.

Any suitable internal standard can be used. For example, the internalstandard can be total creatine when the MRS is based on ¹H resonance, orinternal standard can be adenosine triphosphate (ATP) when the MRS isbased on ³¹P resonance. The term “total creatine” refers to thecombination of creatine and phosphocreatine. Creatine is buffered incell systems; accordingly, the amount of creatine remains substantiallyconstant.

It is contemplated that the method of the present invention isapplicable to monitoring early treatment response wherein the treatmentinvolves inhibition of angiogenesis by interrupting one or more of theproangiogenic pathways involving growth factors, integrins/proteases,coagulation/fibrinolytic factors, and/or inflammatory factors. Inaccordance with an embodiment, the invention provides a method formonitoring early treatment response of a treatment involving the use ofan angiogenesis inhibitor, wherein the angiogenesis inhibitor interruptsa proangiogenic pathway, e.g., one or more pathways selected from thegroup consisting of a growth factor signaling pathway, anintegrin/protease pathway, a coagulation/fibrinolysis pathway, and aninflammatory signaling pathway, and any combination thereof.

Angiogenesis can involve one or more of a number of signaling pathways.Angiogenesis is initiated by hypoxia, mutant oncogenes and/or tumorsuppressor genes that turn on the signaling pathways utilizing signalingproteins. Intracellular organelles are up-regulated to generate theincrease in signaling proteins.

In the growth factors signaling pathway, malignant cells secreteangiogenic growth factors in response to hypoxia, mutated oncogenes,and/or tumor suppressor genes. The proangiogenic growth factors secretedby the tumor react with the normal surrounding cells. Vascularendothelial growth factor (VEGF) plays a role in this signaling pathwayby binding to endothelial cells (EC) and causing their proliferation,increase survival and vascular permeability. Tumor VEGF expression isfurther stimulated by various tumor cytokines and growth factorsincluding basic fibroblastic growth factor (bFGF), platelet derivedgrowth factor (PDGF), insulin-like growth factor (IGF-1), tumor necrosisfactors (TNF-α and TNF-β), angiopoietin-2, as well as hypoxia-induciblefactor-1 (HIF-1). Fisher, et al., supra and Tang, D. G., et al., Semin.Thromb. Hemost. 30, 109-117 (2004). The recruited endothelial cells,fibroblasts, and platelets, in turn, generate growth factors furtherpromoting angiogenesis.

In the integrin/protease signaling pathway, endothelial cells utilizethe surface α₃B₁, integrin receptor to adhere with urokinase-typeplasminogen activator (U-PA) in the extracellular matrix (ECM) to formplasmin, a serine protease. Plasmin dissolves the ECM allowing EC totunnel into the ECM. DeClerck, Y. A., et al., Am. J. Pathol. 161,1131-1139 (2004). Endothelial cells also utilize the surface α_(v)B₃integrin receptor to activate matrix metalloproteinases (MMP) in the ECMallowing the new vessels to grow towards the tumor. Engelse, M. A., etal., Semin. Thromb. Hemost. 30, 71-82 (2004). During angiogenesis theintegrin surface receptors of EC are increased in number by the ECtranscription factors that are stimulated by tumor VEGF. DeClerck etal., supra.

In the coagulation/fibrinolysis signaling pathway, the tissue factor(TF), which is a glycoprotein surface receptor, is the initiator of thesignaling pathway. Usually TF is quiescent, being sequestered on cellswithin the subendothelium of vessel walls. The release of tumor VEGFcauses fenestration of the endothelial layer thereby increasing vascularpermeability and exposing TF. TF comes in contact with circulatingfactor (F) VII that escapes into the ECM because of vascularpermeability. The TF/F VII combination initiates the coagulation cascadeby activating thrombin in the ECM. Thrombin has many proangiogeniceffects including increasing VEGF receptors (VEGFR) on EC, releasingVEGF from platelets, releasing bFGF from ECM and EC, and releasingtissue type plasminogen (tPA) from EC thereby activating MMP to dissolvethe ECM. The final step of the coagulation cascade is cleavage of plasmafibrinogen (FBG) to fibrin by thrombin. Fibrinogen is a plasma proteinof hepatic origin that escapes into the ECM because of vascularpermeability. Proteolytic breakdown of FBG by thrombin releases fibrin.Cross-linked fibrin supports EC adhesion, migration and survival bydegrading bFGF and release of interleukin 8. Fernandez, P. M., et al.,Semin. Thromb. Hemost. 30, 31-44 (2004). Platelets amplify thecoagulation reaction by entering the ECM. The platelets formaggregations and become activated by collagen, thrombin, and fibrinogenin the ECM. The activated platelets release their secretory granulescontaining VEGF, bFGF, IGF-1, PDGF, and Ang-1, as well as otherproangiogenic factors. Sierko, E., et al., Semin. Thromb. Hemost. 30,95-108 (2004).

In the inflammatory signaling pathway, inflammatory cells escape intothe ECM because of vascular permeability. The inflammatory cells adhereto the platelet aggregations in the ECM. The inflammatory cells andplatelets release additional VEGF, as well as, other cytokines thatstimulate angiogenesis, including the arachidonic acid metabolism bycyclooxygenase (COX) and lipoxygenase (LOX) to form prostaglandins andleukotrienes. Nie, D. et al., Semin. Thromb. Hemost. 30, 119-125 (2004).The neoplastic cells also release tumor necrosis factor (TNF),interleukin-1 (IL-1), interleukin-6 (IL-6), and monocytic chemotacticprotein-1, mediating inflammation by attracting macrophages,neutrophils, fibroblasts, and mast cells. The inflammatory cells releasecytokines and chemokines that contribute to angiogenesis. Balkwill, F.,et al., Lancet 357, 539-545 (2001).

In accordance with an embodiment of the invention, the angiogenesisinhibitor can cause an interruption in an up-regulated intracellularorganelle; for example, an interruption of the production of thesecretory granules and/or the transporting vesicles. In accordance withanother embodiment of the invention, the angiogenesis inhibitor cancause an interruption in the function of the Golgi apparatus. In furtherembodiments of the invention, the angiogenesis inhibitor can cause aninterruption in the function of the lysosomes, the endoplasmicreticulum, the mitochondrion, the nucleus, and/or the peroxisomes.

Angiogenesis inhibitors can interrupt the proangiogenic signalingpathways, for example, the growth factor signaling pathway. Productionof growth factors is interrupted by blocking the growth factor cellsurface receptor with tyrosine kinase inhibitors (TKI) or withmonoclonal antibodies. Blackledge, G. et al., Br. J. Cancer 90, 566-572(2004). Examples of VEGF receptor (VEGFR) TKI include SU5416 and SU6668,the latter also inhibiting bFGF and PDGF receptors. Fisher et al.,supra. Monoclonal antibodies to growth factor receptors also inhibitangiogenesis and include, cetuximab, IMC-C225, ABX-EGF, HuMax-EGFR, anda monoclonal antibody to VEGFR (DC101); Fisher et al., supra, andBlackledge et al., supra, as well as other monoclonal antibodies such asgefitinib, erlotinib, canertinib, EKB-569, and lapatinib. Other agentsto block VEGF signaling include VEGF toxin conjugates, dominant negativeVEGF-2, soluble VEGFR (VEGF-TRAP) and antisense oligonucleotides.Suramin, an antiparasitic agent has antiangiogenic effects by inhibitingboth VEGF and bFGF. Fisher et al., supra. Growth factors are alsoinhibited by antagonists of transforming kinases (gleevec, herceptin),famesyl transferase inhibitors, and agents targeting tumor suppressors,such as p53 (PRIMA-1). Yu, J. L. et al., Semin. Thromb. Hemost. 30,21-30 (2004). Examples of angiogenesis inhibitors that inhibit VEGFproliferation of endothelial cells include thalidomide and squalamine.Thalidomide inhibits the processing of messenger RNA (mRNA) for VEGFthereby producing an antiangiogenic effect. Balkwill, F., et al., supra.Squalamine inhibits the VEGF effect by changing the shape of theendothelial cell. Fisher et al., supra.

The angiogenesis inhibitors can inhibit the integrin/protease signalingpathway. The integrin-mediated cell adhesion is interrupted by antibodyand peptide integrin inhibitors, thereby inhibiting angiogenesis.Anti-α_(v)B₃ integrin antibody (Vitaxin), anti-α_(v)B₁, integrinantibody, and a cyclic peptide inhibitor of integrin α_(v)B₃//α_(v)B₅(Cilengitide) are in clinical trials. Jin, H. et al., Br. J. Cancer 90,561-565 (2004). An inhibitor (TNP-470) to urokinase-type plasminogenactivator (U-PA) prevents the EC surface α₃B₁, integrin to adhere,thereby preventing the formation of plasmin. Fisher et al., supra.Inhibitors to plasmin, such as α2-antiplasmin and α2-macroglobulin, haveantiangiogenic activity. Antiangiogenic activity occurs whenkininostatin binds to U-PA receptor (U-PAR), thereby initiatingapoptosis. Colman, R. W., Semin. Thromb. Hemost. 30, 45-61 (2004).Inhibition of matrix metalloproteinases (MPs) occurs by normal tissueinhibitors of MPs (TIMPs). MMP can also be inhibited byα2-macroglobulins. Examples of MMP inhibitors include, BMS275291, COL-3,marimastat, neovastat, and solimastat. Engelse et al., supra and Fisheret al., supra. Angiogenesis inhibitors can be derived from proteolyticcleavage of the basement membrane or from proteolytic cascades.Angiostatin is produced by hydrolysis of plasminogen by MMPs, elastases,and U-PA-activated plasmin and causes endothelial apoptosis. Endostatinis the C-terminal fragment of collagen XVIII and inhibits VEGF inducedmigration of EC, and binds to EC integrins also impairing EC migration.Engelse et al., Semin. Thromb. Hemost. 30, 71-82 (2004).

The angiogenesis inhibitors can inhibit the coagulation/fibrinolyticsignaling pathway. The coagulation/fibrinolytic signaling pathway innormal subjects is quiescent being modulated by proangiogenic andantiangiogenic factors. Several antiangiogenic factors are releasedduring wound healing, and are now being investigated in the treatment ofcancer. During the proteolysis of prothrombin, cryptic kringle fragmentsare released inhibiting angiogenesis. Antithrombin (AT) fragments aregenerated during the proteolytic breakdown of thrombin, blocking TF andVEGF. Fibrinogen-E and fibrin-D fragments both have antiangiogeniceffects. Platelets release thrombospondin-1 and platelet factor-4 (PF-4)both with antiangiogenic effects. The coagulation/fibrinolytic signalingpathway can be blocked with low molecular weight heparins (LMWH) bycompeting with growth factors for binding sites in the ECM.Wojtukiewicz, M. S., et al., Semin. Thromb. Hemost. 30, 145-156 (2004).

The angiogenesis inhibitors can inhibit the inflammatory signalingpathway. Inhibitors to the mobilization of arachidonic acid prevent therelease of angiogenic products including prostaglandin H2 (PGH2) andhydroxyeicosatetraenoic acids (HETEs). Cyclooxygenase (COX) inhibitorsand lipoxygenase (LOX) inhibitors prevent the release of theproangiogenic factors, thereby, blocking the inflammatory signalingpathway and angiogenesis. Nie et al., supra. Examples of COX inhibitorsinclude Vioxx and Celebrex. Balkwill, F., et al., supra. Otherangiogenesis inhibitors in the inflammatory process include interferon-αand β that down-regulate bFGF expression and inhibits its mitogeniceffects. Fisher, et al., supra. Two TNF antagonists (etanercept andinfliximab) have been shown to reduce angiogenesis, prevent leukocyticinfiltration and inhibit MMP. Balkwill, F., et al., supra.

The present invention provides, in an embodiment, a method formonitoring early treatment response as described above, wherein theangiogenesis inhibitor is selected from the group consisting of VEGFreceptor tyrosine kinase inhibitors, monoclonal antibodies to growthfactor receptors, VEGF toxin conjugate, dominant negative VEGF-2,soluble VEGFR (VEGF-TRAP) and antisense oligonucleotides, growth factortransforming kinases, famesyl transferase inhibitors, agents targetingtumor suppressors, agents that inhibit proliferation of endothelialcells, antibody and peptide integrin inhibitors, plasmin inhibitors,urokinase-type plasminogen activator inhibitors, matrixmetalloproteinase inhibitors, cyclooxygenase inhibitors, lipoxygenaseinhibitors, and inhibitors of mitogenic effects, and any combinationthereof.

The present invention provides a method for monitoring early treatmentresponse, wherein the angiogenesis inhibitor is selected from the groupconsisting of SU5416, SU6668, cetuximab, gefitinib, erlotinib,canertinib, EKB-569, lapatinib, IMC-C225, ABX-EGF, HuMax-EGFR, DC101,suramin, gleevec, herceptin, p-53 (PRIMA-1), thalidomide, squalamine,anti-α_(v)B₃ integrin antibody, anti-α_(v)B₅ integrin antibody, cyclicpeptide inhibitor of integrin α_(v)B₃//α_(v)B₅, cilengitide, fumagallin,TNP-470, EMD 121974, α2-antiplasmin, α2-macroglobulin, kininostatin,BMS275291, COL-3, marimastat, neovastat, solimastat, angiostatin,endostatin, antithrombin fragments, fibrinogen-E, fibrin-D,thrombospondin-1, platelet factor-4, low molecular weight heparins,Vioxx, Celebrex, interferon-α and β, and any combination thereof.

MRS is important for evaluating treatment response to cytostaticinhibitors because of the unique information gained. During a single MRSacquisition unique information is gained from the tumor bed and thesurrounding tissue. MRS monitors changes in protein metabolism byobserving the changes in the intracellular membranes of organelles thatgenerate and transport the signaling proteins to the plasma membrane.The changes in the total choline occur before the classical imagingfindings of changes in size and enhancement. Since MRS is a non-invasivestudy, sequential studies can be performed with different inhibitor doseschedules, as well as, with combinations of chemotherapy. Since a web ofsignaling pathways support tumor growth and angiogenesis, a decrease incholine, e.g., total choline, confirms an appropriate inhibitor to tumorgrowth and angiogenesis.

MRS is important for evaluating treatment response because of theavailability of the method. MRS is available commercially on magneticresonance units to study human subjects, animals and cultured, perfusedcellular extracts. MRS can quantify changes in choline, and utilizingsimilar pulse sequences, results can be statistically analyzed fromworldwide sites during drug trials

In accordance with the present invention, any suitable cancer or tumorcan be treated, for example, a cancer selected from the group consistingof brain cancer, colorectal cancer, breast cancer, acute leukemia, lungcancer, kidney cancer, squamous cell cancer, testicular cancer, stomachcancer, melanoma, sarcomas, ovarian cancer, non-small cell lung cancer,esophageal cancer, gastric cancer, pancreatic cancer, neuroblastoma,mesothelioma, prostate cancer, bone cancer, kidney cancer, andhepatocellular cancer.

In accordance with the present inventive method, early treatmentresponse can be measured within a period of about 168 hours, preferablyabout 24 hours, and more preferably about 12 hours, of the treatment.For example, the response can be monitored every 12, 24, 36, 48, 60, 72,84, 96, 108, 120, 132, 144, 156, or 168 hours, or any combinationthereof, after administration of the angiogenesis inhibitor.

The present invention also provides a method for monitoring cancertreatment comprising: (a) localizing a tumor in a patient; (b) selectinga region of interest (ROI) of the tumor and a tissue adjoining orsurrounding the tumor; (c) obtaining magnetic resonance spectra (MRS) ofthe ROI; (d) measuring the amount of Choline, e.g., total choline, fromthe MRS spectra; (e) initiating treatment comprising administering anangiogenesis inhibitor; (f) obtaining MR spectra of the tumor at thesame ROI within a period of 7 days, preferably 3 days, and morepreferably within 1 day, of initiating treatment; (g) measuring theamount of Choline from the MR spectra; and (h) comparing the amount ofCholine obtained before treatment with the amount of Choline obtainedafter treatment; whereby a decrease in the amount of Choline aftertreatment is indicative of a positive response to treatment.

The basis for clinical MR studies (e.g., MRS or MRI) is the one of thenuclei, for example, the hydrogen nucleus—the proton. The same machineryis used for these studies. They differ in the software manipulation ofthe emitted radiofrequency (RF) from the ¹H nuclei. In MRI, the signalis used to create the image; in MRS, the signal is used to create thespectrum. Fourier Transform principle is the basis of the computer thatallows the MRS software to separate the individual RFs within thesignal. The spectrum therefore represents the different RFs beingemitted within the selected region of interest (ROI). The points alongthe horizontal axis of the spectrum represent specific RFs emitted fromeach metabolite. The vertical axis of the spectrum is proportional tothe amount of each metabolite forming the area beneath the RF peaks.Spectra can be obtained on 0.5 to 2.0 T MR scanners, although high-fieldstrength scanners provide better definition of the spectra. Spectraobtained with different-strength scanners can be compared on a scale inparts per million (ppm) along the horizontal axis because metabolitesalways reside at one or more specific sites, for example, alanineresides at 1.47, N-acetylaspartate resides at 2.0 and 2.6 ppm, creatineresides at 3.0 and 3.9 ppm, Choline resides at 3.2 ppm, and waterresides at 5.0 ppm.

Any suitable MR spectrometer can be used in the practice of the presentinvention. Clinical MR spectra can be obtained on MR scanners, forexample, utilizing the clinical spectroscopy package called proton brainexam/single voxel (PROBE/SV) developed by General Electric MedicalSystems (Milwaukee, Wis.) for use with GE's 1.5 Tesla (T) MR scanner.See Norfray, J. et al., supra, and Norfray, J. F. et al., supra, forprocedures for obtaining MR spectra, identification of the peakscorresponding to metabolites such as Choline, creatine, and others, andratio of the peaks. See also Danielsen and Ross, Magnetic ResonanceSpectroscopy Diagnosis of Neurological Diseases, Marcel Dekker, Inc.(1999); Ross, B. et al., Magnetic Resonance Quarterly, 10, 191-247(1994); and Ross et al., U.S. Pat. No. 5,617,861. Based on theinformation in the above publications, as well as information availablein the art, those of skill in the art should be able to practice theinvention on all types of tumors in accordance with the presentinvention.

The present invention can be carried out in any suitable manner, forexample, as follows. Prior to initiating a therapy on a patient, thetumor is localized. Thus, for example, magnetic resonance images (MRI's)of the tumor and the adjoining or surrounding normal tissue, e.g., brainmetastasis, breast malignancy, or bone tumor, with axial, sagittal, andcoronal T1 and T2 images are obtained with and without contrast. Aregion of interest (ROI) is selected. The ROI, in an embodiment, is apart or whole of the tumor and an adjoining or surrounding normal tissuerecruited for angiogenesis. This can be carried out based on the MRIfindings to determine the tumor volume and location to be studied. MRspectra of the ROI are obtained within or outside the tumor utilizingshort and/or long TE (echo time), preferably short echo, pulsesequences. The spectra obtained are interpreted. The peak correspondingto Choline is identified, e.g., at a chemical shift of 3.22 ppm. Basedon the Choline peak, the amount of total cellular membranes isdetermined from either the height of the peak or the area under thepeak. An internal or external standard is identified in the ROI. Anexample of an internal standard is creatine or total creatine. Anexample of an external standard is 100% 2-(trimethylsilyl)-ethanol(TSE), which may be taped to the head coil of the MR spectrometer.Kreis, R. et al., J. Magnetic Resonance, Series B 102, 9-19 (1993). Theratio of the Choline to the standard is calculated. The Choline tocreatine ratio represents a measure of the total cell membranes withinthe ROI of the tumor or outside the tumor prior to treatment.

The treatment of the tumor is initiated by administering an effectiveamount of the angiogenesis inhibitor starting from time zero. The earlytreatment response can be monitored, for example, at 24 hours (day 1) to168 hours (day 7), as follows. The tumor is localized utilizing the sameMRI pulse sequences as prior to the treatment. The same ROI is selectedwithin and/or outside the tumor. MR spectra of the tumor are obtainedutilizing the same pulse sequences, the same TR (relaxation time), TE(echo time), phases, and frequency averages. The MR spectra areinterpreted as before and the Choline to creatine ratios (e.g., heightor area ratios) are calculated.

If the observed decrease in the Choline to creatine ratio is 15% ormore, preferably 20% or more, and more preferably 25% or more relativeto pre-treatment condition, then it can be concluded that an earlyresponse is positive and angiogenesis has been stopped. The earlydecrease in the Choline, e.g., total choline, to creatine ratioidentifies a decrease in the intracellular cell membranes, for example,a decrease in the number of organelles and their granules and/orvesicles. If the ratio of Choline to creatine increases, e.g., a 15% ormore, preferably 20% or more, and more preferably 25% or more, of anearly increase in the ratio is observed, the increase identifies anincrease in the intracellular membranes, for example, an increase in theorganelles and their granules and/or vesicles. A change (decrease) inthe amount of Choline in the region adjoining or surrounding the regionis indicative of inhibition of angiogenesis. A change (decrease) in theamount of Choline in the tumor is indicative of inhibition of the tumor.Thus, MRS can be used to study the early response or effectiveness of acancer treatment, both in inhibiting the tumor itself and in themetastasis of tumor.

The present invention further provides a method for determiningeffectiveness of a molecule as a drug for treating cancer comprisingadministering an amount of the molecule to an animal having a canceroustissue and measuring, by Magnetic Resonance Spectroscopy, the amount ofCholine present in the cancerous tissue before and after administeringthe molecule, wherein the molecule comprises a angiogenesis inhibitor,whereby a decrease in the amount of Choline after administering themolecule is indicative of its effectiveness. The animals that can beused in the present method can be, for example, mammals such as mice,rats, horses, guinea pigs, rabbits, dogs, cats, cows, pigs, monkeys, andhumans. The amount of cell surface receptor inhibitor will vary with anumber of factors, e.g., weight of the animal, type of cancer, andseverity of cancer, and is within the skill of the artisan. Thepotential drug can be administered by any suitable route ofadministration, e.g., oral, aerosol, parenteral, subcutaneous,intravenous, intraarterial, intramuscular, interperitoneal, rectal, andvaginal routes. The cancer can be natural or induced. The effectivenessof a potential drug can be determined within a relatively short periodof time, for example, within 12-168 hours, preferably 12-24 hours.

The present invention further provides a method for monitoring earlytreatment response of an angiogenesis effector treatment in a diseasedanimal comprising measuring, by MRS, the amount of Choline, e.g., totalcholine, present in the diseased tissue before and after treatment,wherein said treatment comprises administration of an angiogenesiseffector, whereby a change in the amount of Choline after treatment isindicative of a positive response. The angiogenesis effector can inhibitangiogenesis or promote angiogenesis. Angiogenesis can be inhibited byinterfering with a proangiogenic signaling pathway involving a growthfactor, integrin/protease, coagulation/fibrinolysis factor, orinflammatory factor. Angiogenesis can be promoted by up-regulating aproangiogenic pathway involving a growth factor, integrin/protease,coagulation/fibrinolysis factor, or inflammatory factor

It is contemplated that the present invention, in an embodiment, can beemployed to monitor the early treatment response of any diseaseinvolving angiogenesis, i.e., non-neoplastic diseases where abnormalangiogenesis contributes to a disease or inducement of angiogenesiscould lead to a treatment of disease, for example, cardiac disease,rheumatoid arthritis, retinopathy, hereditary hemorrhagictelangiecstasia, and hyperplasia. For example, in a patient who had astroke, a growth factor such as the VEGF can be administered to thepatient (e.g., in the brain) and the development of new blood vesselscan be monitored. Further, in cardiac patients, a growth factor such asVEGF, for example, through the use of a catheter to a location in theheart, and the development and growth of new vessels can be monitored.In diseases such as retinopathy and rheumatoid arthritis, the patientundergoing treatment with an angiogenesis inhibitor can be monitored fora decrease in the number or size of blood vessels contributing to thedisease.

The following example further illustrates an embodiment of theinvention, but of course should not be construed as in any way limitingits scope.

Example

This Example illustrates a method for measuring the ratio of Choline tocreatine, in accordance with an embodiment of the invention. Asillustrated in FIG. 1, a region of interest (ROI) and the individualvoxels (V) are used to determine the amount of total choline (orCholine) in cell membranes. An axial T2 FLAIR (Fluid AttenuatedInversion Recovery) image identifies the location of the tumor. An ROIis prescribed to include the tumor, the surrounding white matter, andthe normal contralateral white matter measuring 5 cm×8 cm. Forty spectraare obtained of the entire ROI using multi-voxel technique, with eachvoxel measuring 1 cm×1 cm to reduce partial volume averaging. A specificshort-echo PRESS pulse sequence with a TR 1500 msec, TE 35 msec, 128number of averages, with 24 phase and 24 frequency steps acquires thespectra in 9 minutes and 50 seconds. A software program generatesspectra from selected voxels: V(N)=voxel of normal contralateral whitematter, V(+A)=voxel of white matter undergoing angiogenesis, V(−A)=voxelof white matter not undergoing angiogenesis, V(T)=voxel within thetumor. Segments of the spectra showing the peak heights of total choline(Choline or CHO) and of creatine (CRE) are diagramed for V(N), V(+A),V(−A) and V(T). Choline to creatine ratios are generated (CHO/CRE) toquantify the cell membranes at each location. The V(N) identifies theCHO/CRE=1.0 representing the quantity of normal membranes. At V(+A) theCHO/CRE is increased 20% to 1.2. In V(−A) the CHO/CRE is similar toV(N). In the tumor V(T) the CHO/CRE is increased to 2.0 indicating twicethe amount cell membranes at this location in the tumor.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method for monitoring early treatment response of a cancertreatment comprising measuring, by Magnetic Resonance Spectroscopy(MRS), the amount of Choline present in a tissue adjoining orsurrounding the cancerous tissue before and after treatment, whereinsaid treatment comprises administration of an angiogenesis inhibitor,whereby a decrease in the amount of Choline after treatment isindicative of a positive response.
 2. The method of claim 1, wherein theMRS is based on the resonance of nuclei selected from the groupconsisting of ³¹P, ¹H, ¹³C, and ²³Na, and any combination thereof. 3.The method of claim 2, wherein the MRS is based on ¹H resonance.
 4. Themethod of claim 1, wherein measuring the amount of Choline comprisesmeasuring the height of a peak corresponding to Choline.
 5. The methodof claim 1, wherein measuring the amount of Choline comprises measuringthe area under a peak corresponding to Choline.
 6. The method of claim1, wherein measuring the amount of Choline comprises measuring the ratioof the height of a peak corresponding to Choline relative to the heightof peak of an internal standard.
 7. The method of claim 6, wherein theinternal standard is total creatine when the MRS is based on ¹Hresonance.
 8. The method of claim 6, wherein the internal standard isadenosine triphosphate (ATP) when the MRS is based on ³¹P resonance. 9.The method of claim 1, wherein measuring the amount of Choline comprisesmeasuring the ratio of the area under a peak corresponding to Cholinerelative to the area under a peak of an internal standard.
 10. Themethod of claim 9, wherein the MRS is based on ¹H resonance and theinternal standard is total creatine.
 11. The method of claim 1, whereinthe angiogenesis inhibitor interrupts one or more pathways selected fromthe group consisting of a growth factor signaling pathway, anintegrin/protease pathway, a coagulation/fibrinolysis pathway, and aninflammatory signaling pathway.
 12. The method of claim 1, whereinmeasuring the amount of Choline comprises measuring the amount ofcholine, phosphocholine, phosphatidylcholine, lysophosphatidylcholine,or glycerophosphocholine, phosphomonoesters of choline, phosphodiestersof choline, phosphoethanolamine, glycerophosphoethanolamine, or anycombination thereof.
 13. The method of claim 1, wherein the amount ofCholine is measured within a period of about 168 hours of saidtreatment.
 14. The method of claim 13, wherein the amount of Choline ismeasured within a period of about 24 hours.
 15. The method of claim 14,wherein the amount of Choline is measured within 12 hours of saidtreatment.
 16. The method of claim 1, wherein the angiogenesis inhibitoris selected from the group consisting of VEGF receptor tyrosine kinaseinhibitors, monoclonal antibodies to growth factor receptors, VEGF toxinconjugate, dominant negative VEGF-2, soluble VEGFR (VEGF-TRAP) andantisense oligonucleotides, growth factor transforming kinases, farnesyltransferase inhibitors, agents targeting tumor suppressors, agents thatinhibit proliferation of endothelial cells, antibody and peptideintegrin inhibitors, plasmin inhibitors, urokinase-type plasminogenactivator inhibitors, matrix metalloproteinase inhibitors,cyclooxygenase inhibitors, lipoxygenase inhibitors, and inhibitors ofmitogenic effects, and any combination thereof.
 17. The method of claim1, wherein the angiogenesis inhibitor is selected from the groupconsisting of SU5416, SU6668, cetuximab, gefitinib, erlotinib,canertinib, EKB-569, lapatinib, IMC-C225, ABX-EGF, HuMax-EGFR, DC101,suramin, gleevec, herceptin, p-53 (PRIMA-1), thalidomide, squalamine,anti-α_(v)B₃ integrin antibody, anti-α_(v)B5 integrin antibody, cyclicpeptide inhibitor of integrin α_(v)B₃//α_(v)B₅, cilengitide, fumagallin,TNP-470, EMD 121974, α2-antiplasmin, α2-macroglobulin, kininostatin,BMS275291, COL-3, marimastat, neovastat, solimastat, angiostatin,endostatin, antithrombin fragments, fibrinogen-E, fibrin-D,thrombospondin-1, platelet factor-4, low molecular weight heparins,Vioxx, Celebrex, and interferon-α and β, and any combination thereof.18. The method of claim 1, wherein the cancer is selected from the groupconsisting of brain cancer, colorectal cancer, breast cancer, acuteleukemia, lung cancer, kidney cancer, squamous cell cancer, testicularcancer, stomach cancer, melanoma, sarcomas, ovarian cancer, non-smallcell lung cancer, esophageal cancer, gastric cancer, pancreatic cancer,neuroblastoma, mesothelioma, prostate cancer, bone cancer, kidneycancer, and hepatocellular cancer.
 19. The method of claim 1, whereininhibition of angiogenesis causes an interruption in an up-regulatedintracellular organelle.
 20. The method of claim 19, wherein theinterruption in the up-regulated intracellular organelle decreases thenumber of secretory granules.
 21. The method of claim 19, wherein theinterruption in the up-regulated intracellular organelle decreases thenumber of transporting vesicles.
 22. The method of claim 1, whereininhibition of angiogenesis causes an interruption in a function of theGolgi apparatus.
 23. The method of claim 1, wherein inhibition ofangiogenesis causes an interruption in a function of the lysosomes. 24.The method of claim 1, wherein inhibition of angiogenesis causes aninterruption in a function of the endoplasmic reticulum.
 25. The methodof claim 1, wherein inhibition of angiogenesis causes an interruption ina function of the mitochondrion.
 26. The method of claim 1, whereininhibition of angiogenesis causes an interruption in a function of thenucleus.
 27. The method of claim 1, wherein inhibition of angiogenesiscauses an interruption in a function of the peroxisomes.
 28. A methodfor monitoring cancer treatment comprising: (a) localizing a tumor in apatient; (b) selecting a region of interest (ROI) of the tumor and atissue adjoining or surrounding the tumor; (c) obtaining magneticresonance spectra (MRS) of the ROI; (d) measuring the amount of Cholinefrom the MRS spectra; (e) initiating treatment comprising administeringan angiogenesis inhibitor; (f) obtaining MR spectra of the tumor at thesame ROI within a period of 7 days of initiating treatment; (g)measuring the amount of Choline from the MR spectra; and (h) comparingthe amount of Choline obtained before treatment with the amount ofCholine obtained after treatment; whereby a decrease in the amount ofCholine after treatment is indicative of a positive response totreatment.
 29. The method of claim 28, wherein the MR spectra of thetumor is obtained within 3 days of initiating treatment.
 30. The methodof claim 29, wherein the MR spectra of the tumor is obtained within 1day of initiating treatment.
 31. A method for determining effectivenessof a molecule as a drug for treating cancer comprising administering anamount of the molecule to an animal having a cancerous tissue andmeasuring, by Magnetic Resonance Spectroscopy, the amount of Cholinepresent in the tissue surrounding the cancerous tissue before and afteradministering the molecule, wherein said molecule comprises anangiogenesis inhibitor, whereby a decrease in the amount of Cholineafter administering said molecule is indicative of its effectiveness.32. A method for monitoring early treatment response of an angiogenesiseffector treatment in a diseased animal comprising measuring, byMagnetic Resonance Spectroscopy (MRS), the amount of Choline present inthe diseased tissue before and after treatment, wherein said treatmentcomprises administration of an angiogenesis effector, whereby a changein the amount of Choline after treatment is indicative of a positiveresponse.
 33. The method of claim 32, wherein the angiogenesis effectoris an inhibitor of angiogenesis.
 34. The method of claim 32, wherein theangiogenesis effector is a promoter of angiogenesis.
 35. The method ofclaim 33, wherein the disease is retinopathy, rheumatoid arthritis,hereditary hemorrhagic telangiecstasia, or hyperplasia.
 36. The methodof claim 34, wherein the disease is a stroke or cardiac disease.
 37. Themethod of claim 33, wherein angiogenesis is inhibited by interferingwith a proangiogenic signaling pathway involving a growth factor,integrin/protease, coagulation/fibrinolysis factor, or inflammatoryfactor.
 38. The method of claim 34, wherein angiogenesis is promoted byup-regulating a proangiogenic pathway involving a growth factor,integrin/protease, coagulation/fibrinolysis factor, or inflammatoryfactor.
 39. The method of claim 1, wherein the Choline is the totalcholine.
 40. The method of claim 28, wherein the Choline is the totalcholine.
 41. The method of claim 31, wherein the Choline is the totalcholine.
 42. The method of claim 32, wherein the Choline is the totalcholine.